COMPARATIVE A peer-reviewed open-access journal CompCytogen 6(3): 323–333Cytogenetic (2012) analysis in Thoracocharax stellatus (Kner, 1858)... 323 doi: 10.3897/CompCytogen.v6i3.3637 RESEARCH ARTICLE Cytogenetics www.pensoft.net/journals/compcytogen International Journal of Plant & Animal Cytogenetics, Karyosystematics, and Molecular Systematics Cytogenetic analysis in Thoracocharax stellatus (Kner, 1858) (Characiformes, Gasteropelecidae) from Paraguay River Basin, Mato Grosso, Brazil Edson Lourenço da Silva1, Rafael Splendore de Borba1, Liano Centofante2, Carlos Suetoshi Miyazawa3, Patrícia Pasquali Parise-Maltempi1 1 Instituto de Biociências, UNESP Univ Estadual Paulista, Campus de Rio Claro, Departamento de Biologia, Laboratório de Citogenética. Av. 24A, 1515. CEP: 13506-900. Rio Claro, SP, Brazil 2 Instituto de Biociên- cias, UFMT Univ Federal de Mato Grosso, Departamento de Biologia e Zoologia. Laboratório de Citogenética Animal. Av. Fernando Corrêa da Costa s/n, CCBS-II, CEP: 78060-900. Cuiabá, MT, Brazil 3 Universidade Federal do ABC, Centro de Ciências Naturais e Humanas (CCNH). Rua Santa Adélia, 166. Bairro Bangu, CEP 09.210-170. Santo André, SP, Brazil Corresponding author: Patrícia Pasquali Parise-Maltempi ([email protected]) Academic editor: G. Furgala-Selezniow | Received 6 July 2012 | Accepted 11 September 2012 | Published 26 September 2012 Citation: Silva EL, Borba RS, Centofante L, Miyazawa CS, Parise-Maltempi PP (2012) Cytogenetic analysis in Thoracocharax stellatus (Kner, 1858) (Characiformes, Gasteropelecidae) from Paraguay River Basin, Mato Grosso, Brazil. Comparative Cytogenetics 6(3): 323–333. doi: 10.3897/CompCytogen.v6i3.3637 Abstract Thoracocharax stellatus (Characiformes, Gasteropelecidae) is a small Neotropical species of fish, widely dis- tributed in several rivers of South America. Evidence for karyotype heteromorphysm in populations from different geographical regions has been reported for this species. In this way, populations of T. stellatus from the Paraguay River basin were cytogenetically characterized and the results were compared with oth- er studies performed in the same species but from different basins. The results showed a diploid number of 2n = 54 for T. stellatus, with chromosomes arranged in 6 metacentric (m), 6 submetacentric (sm), 2 sub- telocentric (st) and 40 acrocentric (a), for both sexes, with a simple Nucleolus Organiser Region (NOR) system reported by the techniques of silver nitrate impregnation and fluorescent in situ hybridisation (FISH) using 18S rDNA sequences as probe. The distribution of constitutive heterochromatin, observed by the C-band technique and Chromomycin A3 staining showed great similarity among the analyzed populations and consists mainly of discrete blocks in the pericentromeric and telomeric regions of most chromosomes. The presence of female heterogamety was also observed indicating a ZZ/ZW system with W chromosome almost totally heterochromatic. The results also show cytogenetic diversity of the group and are useful to understand the mechanisms of karyotype evolution of the family. Copyright Edson Lourenço da Silva et al. This is an open access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC-BY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 324 Edson Lourenço da Silva et al. / Comparative Cytogenetics 6(3): 323–333 (2012) Keywords Thoracocharax stellatus, Ag-NOR, C-band, FISH, 18S rDNA Introduction The family Gasteropelecidae (Characiformes) comprises a group of small Neotropical fishes that inhabit rivers of the main Central and South America basins, with exception of the southeast of Brazil and Chile (Géry 1977, Weitzman and Palmer 2003). Three genera are recognized in this family (Carnegiella Eigenmann, 1909, Gasteropelecus Sco- poli, 1777 and Thoracocharax Fowler, 1906), comprising a total of nine nominal species. The first taxonomic study regarding this family was conducted by Weitzman (1954, 1960) which suggested, by osteological observation, the genus Thoracocharax represents an independent lineage inside the group, named as tribe Thoracocharacini. The genera Gasteropelecus and Carnegiella comprise a sister group belonging to a sec- ond lineage named tribe Gasteropelecini (Weitzman 1960). Despite the reduced number of species, this family presents many taxonomic prob- lems related to the difficulty of finding species-specific characters. Carnegiella marthae (Myers, 1927) e.g. have osteological differences related to anal fin rays among speci- mens from Orinoco and Negro rivers, and the specimens from Peruvian Amazon and Madeira Rivers (Géry 1977). So the typical form from Orinoco-Negro has been called Carnegiella marthae marthae, and the species from Peruvian Amazon called Carnegiella marthae schereri (Géry 1977). Thus, considering its large distribution, an accurate analysis of specimens from dif- ferent localities can reveal the existence of putative new species (Weitzman and Weitz- man 1982, Weitzman and Palmer 1996, Weitzman and Palmer 2003). As regards to the cytogenetic aspects, the family Gasteropelecidae is relatively non-conserved, with diploid numbers varying from 2n=48 to 2n=54. The species studied until now are recognized by the presence of many subtelocentric and ac- rocentric chromosomes and almost all presenting one chromosome pair carrier the NOR, that can be variable only in some populations (Carvalho et al. 2002). Also been described and characterized in the family the sexual system ZZ/ZW for the genera Carnegiella and Thoracocharax. The genus Thoracocharax is the most widely distributed among the gasteropelecids, and is characterized by species that have distinct chromosomal and morphological characteristics in different geographic regions. Thoracocharax stellatus(Kner, 1858), for example, presents a considerable morphological polymorphism among populations, mainly due to the geographic isolation (Silva et al. 2009) The karyotypic diversity observed in T. stellatus comprises different diploid num- bers and polymorphism in NOR (Nucleolus Organiser Region) number and different chromosomes involved in the sexual differentiation (ZZ/ZW system) (Carvalho et al. 2002, Venere et al. 2007). These variations can help determining the taxonomic state of some populations. Cytogenetic analysis in Thoracocharax stellatus (Kner, 1858)... 325 Due to the cytogenetic diversity already observed for different T. stellatus popula- tions, the objective of this study was to describe the karyotypic structure of three popula- tions from the Paraguay basin not studied yet and identify chromosomal markers for this species by establishing chromosomal variation patterns among different populations. Material and methods For the cytogenetic analysis of T. stellatus, 111 specimens collected in the Paraguay basin were used, 38 from Cuiabá River - São Gonçalo (SG) (15°39'9.96"S, 56°4'8.62"W), 26 in Cuiabá River - SESC (SE) (16°38'55.0"S, 56°28'06.2"W) and 47 from Barra do Bugres (BB) (15°4'41.13"S, 57°10'55.64"W) (Fig. 1). The material collected was deposited in the Laboratório de Citogenética Animal/UFMT, Mato Grosso (LCA 018, LCA 023, LCA 028). Direct cytological preparations were produced from kidney samples according to the methods given by Foresti et al. (1981). Karyotype analysis was conducted after conventional staining with Giemsa. The chromosomes were morphologically classified according to the nomenclature proposed by Levan et al. (1964), with the following modifications: metacentric (m), submetacentric (sm), subtelocentric (st), and acrocentric (a). Heterochromatic re- gions were visualized by C-banding (Sumner 1972) and double fluorochrome staining (Chromomycin A3 + 4,6 – diamide 2 phenylindole - DAPI), according to the methods used by Schweizer (1980) and NORs were detected by silver nitrate impregnation (Ag- NORs), according to the procedure described by Howell and Black (1980). Figure 1. Sampling sites of the three populations of T. stellatus. SG: São Gonçalo; SE: SESC and BB Barra do Bugres. 326 Edson Lourenço da Silva et al. / Comparative Cytogenetics 6(3): 323–333 (2012) The Fluorescentin situ hibridization (FISH) analyses were performed basically according to the method of Pinkel et al. (1986) with some modifications according to Silva et al. (2012). The 18S rDNA probe was obtained by PCR using specific primers set (NS1 5’-GTAGTCATATGCTTGTCTC-3’ and NS2 5’-GGCTGCTGGCAC- CAGACTTGC-3’) and labelled with digoxigenin by PCR. Metaphase chromosome slides were incubated with RNAse (40 μg/ml) for 1 h at 37°C and dehydrated using an ethanol series (70%, 85%, 100%). Chromosomal DNA was denatured for 1 min and 45 seconds in 70% formamide/2× SSC at 70°C and the spreads were dehydrated using the same ethanol series. The hybridisation solution (50% formamide/2× SSC, 10% dextran sulfate, and 1.5 μg/ml DNA probe) was denatured for 10 min at 95°C and applied to each slide under a coverslip. The hybridisation was performed overnight at 37°C in a moist chamber containing 2× SSC. Post-hybridisation washes were con- ducted using: 50% formamide/2× SSC (pH 7.0) twice for 5 min at 45°C; 2× SSC at 45°C; and 2× SSC at room temperature. Signals were detected using antidigoxigenin- rhodamine antibody. Chromosomes were counterstained with DAPI (1.5 μg/mL) and mounted in antifading solution. Chromosomes were observed using an Olympus BX51 microscope coupled to an Olympus digital camera model D71. Chromosome images were captured using the